Functional characterization of Cullin-1-RING ubiquitin ligase (CRL1) complex in Leishmania infantum

Cullin-1-RING ubiquitin ligases (CRL1) or SCF1 (SKP1-CUL1-RBX1) E3 ubiquitin ligases are the largest and most extensively investigated class of E3 ligases in mammals that regulate fundamental processes, such as the cell cycle and proliferation. These enzymes are multiprotein complexes comprising SKP1, CUL1, RBX1, and an F-box protein that acts as a specificity factor by interacting with SKP1 through its F-box domain and recruiting substrates via other domains. E3 ligases are important players in the ubiquitination process, recognizing and transferring ubiquitin to substrates destined for degradation by proteasomes or processing by deubiquitinating enzymes. The ubiquitin-proteasome system (UPS) is the main regulator of intracellular proteolysis in eukaryotes and is required for parasites to alternate hosts in their life cycles, resulting in successful parasitism. Leishmania UPS is poorly investigated, and CRL1 in L. infantum, the causative agent of visceral leishmaniasis in Latin America, is yet to be described. Here, we show that the L. infantum genes LINF_110018100 (SKP1-like protein), LINF_240029100 (cullin-like protein-like protein), and LINF_210005300 (ring-box protein 1 –putative) form a LinfCRL1 complex structurally similar to the H. sapiens CRL1. Mass spectrometry analysis of the LinfSkp1 and LinfCul1 interactomes revealed proteins involved in several intracellular processes, including six F-box proteins known as F-box-like proteins (Flp) (data are available via ProteomeXchange with identifier PXD051961). The interaction of LinfFlp 1–6 with LinfSkp1 was confirmed, and using in vitro ubiquitination assays, we demonstrated the function of the LinfCRL1(Flp1) complex to transfer ubiquitin. We also found that LinfSKP1 and LinfRBX1 knockouts resulted in nonviable L. infantum lineages, whereas LinfCUL1 was involved in parasite growth and rosette formation. Finally, our results suggest that LinfCul1 regulates the S phase progression and possibly the transition between the late S to G2 phase in L. infantum. Thus, a new class of E3 ubiquitin ligases has been described in L. infantum with functions related to various parasitic processes that may serve as prospective targets for leishmaniasis treatment.


Introduction
Leishmania infantum and L. donovani are the etiological agents of visceral leishmaniasis (VL), the most severe form of leishmaniasis, with fatality rates of up to 95% if not treated.One billion people worldwide live in regions endemic to leishmaniasis, with an estimated 50,000-90,000 new VL cases per year globally, most of which occur in Brazil, East Africa, and India [1].Leishmania has a dixenous lifestyle that comprises an extracellular stage, the promastigote form, which replicates in the gut of sandflies over two replicative phases that consist of various differentiation stages, and an intracellular stage, the amastigote form, which replicates within mammalian macrophages [2,3].This intricate life cycle underscores the importance of understanding the molecular mechanisms governing their survival and replication under diverse conditions.As Leishmania infections continue to pose a serious health problem worldwide, it is critical to unravel the molecular machinery of the parasite.In this regard, investigation of the ubiquitin-proteasome system (UPS) has emerged as a promising avenue, considering its pivotal role in regulating various cellular processes.The UPS orchestrates targeted protein degradation, regulating important aspects of parasite biology [4][5][6][7][8].
Proteasomes are multi-subunit, multicatalytic proteases responsible for most of intracellular proteolysis in eukaryotes [9].L. mexicana proteasome is similar to proteasomes from other eukaryotes [10] and its inhibition has been explored as a potential target for chemotherapy of leishmaniasis [5,7].Lactacystin, a proteasome-specific inhibitor, suppressed the growth of L. infantum promastigotes in vitro and impaired the survival of amastigotes in infected host cells when the promastigotes were previously treated with the inhibitor [11].These findings demonstrate that the UPS is required for parasite replication and amastigote survival within the host cells.
The UPS comprises of three classes of enzymes responsible for protein ubiquitination and the 26S proteasome.Initially, ubiquitin-activating enzyme (E1) catalyzes ubiquitin activation in an ATP-dependent reaction.Furthermore, activated ubiquitin (Ub) is transferred to the ubiquitin-conjugating enzyme (E2), and ubiquitin ligases (E3), which specifically interact with substrates, recruit E2-Ub, culminating in ubiquitination of the substrate [12], which can be reversed by deubiquitinating enzymes (DUBs).E3 ubiquitin ligases are classified into two main classes based on their E2 interaction domains: HECT (Homologous to E6AP Carboxyl Terminus), which directly catalyzes the covalent attachment of ubiquitin to substrate proteins, and RING type (Really Interesting New Gene), which is characterized by their RING or RING-like (e.g., U-box) catalytic domain that promotes Ub transfer from an E2-Ub to the substrate [13].There are approximately 30 E3 HECT-like ligases in mammals and more than 600 RING-like E3 ligases have been predicted in the human genome [14].RING-type E3 ubiquitin ligase members can function as monomers, dimers, and heterodimers or can be multi-subunits based on the Cullin protein, which includes Cullin-1 RING ubiquitin ligases (CRL1s), also called the SCF1 complex (SKP1, CUL1, RBX1, and F-box) [13].CUL1 is a scaffold protein for the RING box protein 1 (RBX1) at the C-terminus and SKP1 at the N-terminus.It is an adapter for F-box proteins (FBP) that uses the F-box motif to interact with SKP1 and other domains to recruit their substrates for the CRL1 complex.Thus, FBPs are specificity factors of CRL1 or SCF1 E3 ubiquitin-ligase complexes [15][16][17].
Two E1 enzymes have been described in L. major [18], 13 E2 enzymes, 79 E3 ubiquitin ligases [19], and 20 deubiquitinating enzymes (DUBs) have been described in L. mexicana [20].The E2 enzymes UBC1/CDC34, UBC2, UEV1, and E3 HECT2 are required for the differentiation of promastigotes to amastigotes.The E1 enzyme UBA1b, E2 enzymes UBC9, UBC14 and E3 enzymes HECT7 and HECT11 are required for proliferation during infection in mice [19].In L. major, Kin13 protein is degraded by the parasite UPS, and its endogenous levels depend on the cell cycle [21].In L. donovani, degradation of pteridine reductase 1 (PTR1) has been suggested to be related to the peptide 63 QADLSNVAK 71 and lysine residue 156 (K156) as probable ubiquitination site, suggesting that the proteasome is the main regulator of its level in the parasite [22].Three proteasome inhibitors, MG132, lactacystin, and epoxomycin, lead to the accumulation of methionine adenosyltransferase (MAT) protein in parasites.Additionally, MAT was conjugated with ubiquitin molecules, suggesting that the UPS was responsible for the degradation of this substrate [23].
CRLs E3 ubiquitin ligases are responsible for eukaryotic cell cycle regulation via ubiquitination of different substrates [24].Despite their well-established roles in cellular processes in various organisms, the involvement of CRLs in Leishmania remains unexplored.In this study, we unveiled the identity of CRL1 components in L. infantum and identified by mass spectrometry the LinfSkp1 and LinfCul1 protein partners involved in different cellular processes, including the UPS.We showed that CRL1 is assembled in L. infantum in association with six Fbox proteins, collectively known as F-box-like proteins (Flp).Through in vitro ubiquitination assays, we provided compelling evidence demonstrating the ability of LinfCRL1(Flp1) to transfer ubiquitin.Finally, gene knockouts of LinfCRL1 components revealed that LinfSKP1 and LinfRBX1 are critical genes, and that LinfCUL1 is involved in cellular proliferation, rosette formation and cell cycle regulation in L. infantum promastigotes.

Identification of CRL1 genes in Leishmania spp
The CRL1 complex comprises CUL1 as a scaffold protein to allow interaction with SKP1 at its N-terminus and RBX1 at its C-terminus.SKP1, in turn, interacts with an F-box protein at different binding interfaces that interact with CUL1 (Fig 1A).Orthologous genes corresponding to H. sapiens SKP1, CUL1, and RBX1 were searched in L. infantum genome using TriTrypDB resources [25].The genes LINF_110018100 (SKP1-like protein), LINF_240029100 (cullin-like protein-like protein), and LINF_210005300 (ring-box protein 1 -putative) were retrieved, and their translated sequences displayed 63, 21 and 67% amino acid identity with their respective human orthologs, respectively.These orthologs were also identified in other Leishmania species, such as L. donovani, L. major, L. mexicana, and L. braziliensis, with a similar identity degree observed in L. infantum, demonstrating that these proteins are conserved in these parasites (Fig 1B -1D).
Guided by the crystallographic structure of human CRL1 [17,26], we identified crucial residues for complex CRL1 assembly in L. infantum (color pattern shown in Fig 1A).Conserved amino acid residues of SKP1/CUL1 and CUL1/RBX1 interaction regions were observed in Leishmania (highlighted by asterisks, Fig 1B -1D).Although LinfCul1 has a low amino acid identity (21%) with its human ortholog, essential residues for its interaction with LinfSkp1 and LinfRbx1 are highly conserved (Fig 1C, green and purple asterisks, respectively).LinfSkp1 residues that are important for interaction with the F-box domain and LinfCul1 are also conserved (Fig 1B, red and black asterisks, respectively).The RING box domain of LinfRbx1 (residues 53-98), which mediates interaction with E2, is also highly conserved in Leishmania (Fig 1D, blue asterisks).These findings indicate that the residues responsible for the assembly of LinfCRL1 are conserved in Leishmania, suggesting its presence in these parasites.

Phylogenetic analysis of CRL1 genes in Leishmania
Since the genome of Leishmania spp.are largely conserved, it is plausible to assume that genes that code for proteins of the CRL1 complex exist in other species of the Leishmania genus.Thus, we expanded our in silico search of the CRL1 complex to include 12 species.Blastx searches in TriTrypDB using human SKP1, CUL1, and RBX1 sequences retrieved the orthologous of L. braziliensis, L. panamensis, L. amazonensis, L. mexicana, L. donovani, L. infantum, L. aethiopica, L. tropica, L. major, L. gerbilli, L. arabica, and L. turanica.We assessed phylogenetic relationships in each reconstructed tree for each member of the CRL1 complex.
Multiple sequence alignment (MSA) obtained through MUSCLE for each protein was verified for the amino acid substitution model, in which LG [29], JTT [30] and JTT with gamma distribution were the most suitable for SKP1, CUL1, and RBX1, respectively.Maximum Likelihood (ML) and Bayesian Inference (BI) methods were then used to reconstruct the phylogenetic trees for each protein group.Overall, the phylogenetic relationships among species based on the reconstructions of the orthologous SKP1, CUL1, and RBX1 proteins (Fig 2) resemble phylogenomic analysis using approximately 5,600 orthologous proteins of trypanosomatids [31].As expected, in all phylogenies, L. infantum forms a clade with L. donovani within the Leishmania subgenus, as does L. mexicana with L. amazonensis, and L. braziliensis with L. panamensis within the Viannia subgenus.
Evolutionary relationships with highest statistical support (bootstrap > 70% and probability < 0.05) were given remarkably to CUL1 phylogeny (Fig 2A) and RBX1 phylogeny (Fig 2B).Bayesian-inferred phylogenies for CUL1 and RBX1 were displayed in S1 Fig For SKP1, most of the branches were not well-supported in phylogeny inferred by the ML method

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Cullin-1-RING ubiquitin-ligase in Leishmania infantum (Fig 2C ), except for the clade formed by species of Viannia subgenus (L.braziliensis and L. panamensis).In turn, Bayesian inference reconstructed a statistically robust tree for SKP1 phylogeny (Fig 2D ), including a better placement of the clade formed by L. mexicana and L. amazonensis, which was inconsistent in the ML-inferred tree.Overall, the phylogeny of the large multidomain CUL1 protein (796AA) proved to be highly conserved, most likely because of its key role as a scaffold in the CRL1 assembly complex.mexicana (LmxM.21.0023) and L. braziliensis (LbrM.21.0040).Blue asterisks indicate the RING domain of RBX1.MSAs were performed using the MUSCLE method [27] and visualized using the ESPript 3.0 [28].The highlighted boxes indicate the conserved regions across the protein sequences.https://doi.org/10.1371/journal.ppat.1012336.g001).The proteins were aligned using the MUSCLE method [27] and trees were reconstructed using ML and BI methods.A total of 183, 744, and 106 positions for SKP1, CUL1, and RBX, respectively, were considered for the final dataset.ML analysis was performed using MEGA11 software [32], while BI analysis was performed using BEAST software [33].The numbers next to the branches represent the statistical support of phylogenetic inference displayed by method side by side, IB (probability) / ML (boostrap), for CUL1 (A) and RBX1 (B); for SKP1 cladograms are displayed according to the method of inference (C and D).Species within Leishmania and Viannia subgenera are indicated in blue and red font, respectively.https://doi.org/10.1371/journal.ppat.1012336.g002

Protein interaction assays with LinfSkp1, LinfCul1 and LinfRbx1
To investigate whether the conserved interaction regions of the L. infantum CRL1 (LinfCRL1) components are sufficient to promote the assembly of this complex, we evaluated by co-immunoprecipitation (co-IP) the interaction among the parasite proteins and their respective partners of H. sapiens (Hs).Initially, HEK293T cells were co-transfected with LinfSkp1-HA and a human protein containing the F-box domain (2xFLAG-FBXO7) (Hs), which interacts with SKP1(Hs) [34].After co-IP, we observed that these proteins interacted, suggesting that the interaction region of LinfSkp1 with the F-box domain is conserved (Fig 3A).Similarly, LinfSk-p1-HA and CUL1-FLAG (Hs) interacted, indicating that LinfSkp1 also possesses a conserved interaction domain with CUL1 (Fig 3B ) in addition to the F-box interaction domain (Fig 3A).To evaluate the LinfCul1 interaction regions with SKP1 and RBX1, HEK293T cells were cotransfected with LinfCul1-FLAG, SKP1-HA (Hs), and RBX1-myc (Hs).Co-IP results demonstrated that LinfCul1 interacted with both proteins (Fig 3C).Lastly, we assessed whether LinfRBX1 interacts with CUL1 (Hs) (Fig 3D).The results demonstrated that both proteins interact, reinforcing the significant conservation of the interaction domain between these proteins, as shown by in silico analyses.Finally, to assess whether LinfCRL1 proteins can assemble a CRL1 complex, co-IP among LinfSkp1-FLAG, LinfCul1-myc, and LinfRbx1-HA was performed, and the results showed that these proteins were co-eluted, demonstrating that they assemble a LinfCRL1 complex in L. infantum (Fig 3E).

LinfCRL1 shares structural similarities with the H. sapiens complex
To gain insight into the structure of LinfCRL1 compared to H. sapiens, we searched in Alpha-Fold database [35,36] for the structures of proteins encoded by the genes LINF_110018100 (LinfSkp1), LINF_240029100 (LinfCul1), and LINF_210005300 (LinfRbx1) of L. infantum JPCM5 and compared them individually to their human orthologs (S2 Fig) .These findings demonstrated a comparable folding pattern of these proteins.The root-mean-square distance (RMSD), a quantitative measure assessing the disparity between corresponding atoms in two structures, confirmed a high similarity between SKP1 proteins with an RMSD value of 1.7 Å.For identical structures, the RMSD is 0, and values within the range of 0-2 Å indicate a high percentage of identity [37].The RMSD values for CUL1 and RBX1 were 4.8 Å and 7 Å, respectively, indicating that although these proteins are not identical, they exhibit notable structural resemblances.
To analyze the LinfCRL1 complex structure, AlphaFold was used to assemble these proteins in a complex using AlphaFold multimer [38].Interestingly, structural models generated showed highly similar topology and folding profiles for both complexes (Fig 4A and 4B).Linf-Cul1 function as a scaffold for the LinfCRL1 complex, interacting with LinfSkp1 at the N-terminus and LinfRbx1 at the C-terminus.The upper view of the complex superimposition from both organisms (Fig 4C, bottom image) revealed a twist in LinfCul1 compared with H. sapiens CUL1.This could be explained by the low identity with the human protein in its central region (Fig 1B) and the lower confidence of its N-terminus observed in the structure predicted by AlphaFold (S2 Fig) .However, the model returned the same structural pattern for the binding of LinfRbx1 to LinfCul1 at the C-terminus ends as the H. sapiens complex (Fig 4C).
Reliability of the LinfCRL1 complex was evaluated using AF2 prediction.The model obtained for the complex contained 1,133 residues and was predicted with an average pLDDT of 81,62, indicating a complex model with good confidence for individual structures and the complex [38].The complex was colored by AF2 using a pLDDT scale, which indicated that most portions of the model were highly reliable, with pLDDT over 80.0, and the less reliable fragments were restricted to mobile fragments at the C-and N-termini of individual proteins

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Cullin-1-RING ubiquitin-ligase in Leishmania infantum (S3 Fig).Thus, the LinfCRL1 complex model not only demonstrated significant structural resemblance to the CRL1 complex from H. sapiens, but also exhibited high reliability in each component and the predicted complex, indicating a conserved function in substrate ubiquitination.

Interactome of LinfSkp1 and LinfCul1
To confirm the assembly of LinfCRL1, the interactome of LinfSkp1 and LinfCul1 was carried out.CRISPR-Cas9 technology was used to generate N-terminally tagged 3x myc-mCherry:: LinfCUL1 or 3x myc-mCherry::LinfSKP1 lines in a transgenic line of L. infantum HU-UFS14 expressing Cas9 and T7 RNA polymerase as previously described [39].The transgenic lines were confirmed by diagnostic PCR using specific primers targeting the integrated cassette (S4A Fig) .These lines showed no difference in their respective duplication rates when compared to the L. infantum-Cas9 T7 parental strain or L. infantum wild-type parasite (S4B Fig) .Proteins from these strains were extracted and subjected to immunoprecipitation (IP) with anti-myc conjugated beads, and the results showed that 3xmyc-mCherry-LinfSkp1 and 3xmyc-mCherry-LinfCul1 were expressed and purified (Fig 5A).

Cullin-1-RING ubiquitin-ligase in Leishmania infantum
To obtain the interactome of LinfSkp1, LinfCul1, the parental strain Cas9 T7, and L. infantum strains were subjected to large-scale IP with anti-myc beads.After washing, the beads were directly digested by trypsin, aiming to increase peptides recovery, and were processed for mass spectrometry analysis [40].The proteins identified in the negative control were excluded from the LinfSkp1 and LinfCul1 interactomes, and a total of 43 unique proteins were identified in the LinfSkp1 interactome and 22 in the LinfCul1 interactome (Fig 5B and S2 Table).Based on the number of unique peptides, LinfCul1 had the highest score in the LinfSkp1 interactome, and LinfRbx1 was identified.LinfSkp1 was the second most abundant protein in the LinfCul1 interactome, which also identified LinfRbx1 (S2 Table and Fig 5B).These results confirm the assembly of the LinfCRL1 complex.To validate the interaction between LinfSkp1 and Linf-Cul1, a transgenic line expressing HA-LinfSkp1 and 3x myc-mCherry-LinfCul1 was generated (S4C Fig) .Promastigote parasites were lysed and subjected to IP with anti-myc beads, and the eluate confirmed the interaction between both proteins in L. infantum (Fig 5C).
In addition to SKP1, CUL1, and RBX1, active CRLs comprise an F-box protein that binds to SKP1 and recruits the substrate for ubiquitination [11,12].Interestingly, five F-box proteins were identified in the LinfSkp1 interactome and two were identified in the LinfCul1 interactome (Fig 5B).Other proteins identified in the LinfSkp1 and LinfCul1 interactomes were UPS members (9 proteins), including a ubiquitin-like protein, as well as proteins related to different cellular processes, such as phosphatases (2), kinases (3), nucleic acid binding ( 9), cilium and flagellum-related proteins (2), ribosomal proteins (6), metabolism-related proteins (1), RNA processing (1), intracellular transport (1), and proteins of unknown function ( 18) (Fig 5B).These results confirmed the presence of an active CRL1 complex in L. infantum and further supported the role of LinfSkp1 and LinfCul1 in fundamental parasitic processes.

F-box proteins associated with functional LinfCRL1 complex
F-box proteins contain a conserved motif of 48 amino acid residues for interaction with SKP1 and a substrate interaction domain, ensuring the specificity of ubiquitination by CRL1 complexes.In humans, there are approximately 69 F-box proteins that play a central role in regulating various cellular processes such as proliferation, cell cycle progression, transcription, and apoptosis [41].Consequently, dysregulation of the ubiquitination process due to overexpression, low expression, or mutation of these proteins leads to several human diseases, including cancer [24].The search for F-box domains in L. infantum proteins using InterPro (IPR036047 -F-box-like domain superfamily) or ortholog genes in Uniprot and TritrypDB identified 11 proteins with an F-box domain in this parasite, of which six were identified in the LinfSkp1 and LinfCul1 interactomes (Table 1).The alignment of the F-box consensus sequence [41] with these six proteins, identified a conservation of the main residues involved in the interaction with SKP1 (Fig 6A ).
Although F-box proteins have been described in trypanosome genomes [42], no studies on this protein group have demonstrated its function in Leishmania spp.Therefore, to elucidate the function of genes termed hypothetical or unknown in Leishmania spp.genomes, we proposed classifying the F-box proteins identified in this study as F-box-like proteins (Flp) (Table 1).To validate the interactions of LinfFlp1, LinfFlp2, LinfFlp3, LinfFlp5 and LinfFlp6 with LinfSkp1, co-IP assays were performed in HEK293T cells transfected with plasmids encoding these genes.The eluates revealed the interaction of these LinfFlps with LinfSkp1 (Fig 6B ), confirming the interactome results.The proteins LinfFlp1 (LINF_240015400) and LinfFlp2 (LINF_150014400) were identified as the second and third proteins in the LinfSkp1 interactome, respectively, with the highest number of sequenced peptides, only behind Linf-Cul1, which is a partner of LinfSkp1 in the CRL1 complex (Fig 5C).Since the number of

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Cullin-1-RING ubiquitin-ligase in Leishmania infantum observed peptides has been utilized to ascertain the abundance of F-box proteins associated with CRL complexes [43], our findings indicate a strong association of LinfFlp1 and LinfFlp2 with LinfSkp1, suggesting a function of LinfCRL1(Flp1) and LinfCRL1(Flp2) in L. infantum.
LinfFlp2 was identified in the LinfCul1 interactome, with the highest number of peptides, followed by LinfSkp1 (S2 Table ).We speculate that this interaction is a result of LinfCul1 interaction with LinfSkp1, which indirectly brings LinfFlp2 from the functional complex.In fact, LinfFlp4, which only appeared in the LinfCul1 interactome, did not directly interact with Linf-Cul1, as demonstrated by the co-IP results (S4D Fig).However, LinfFlp4 interacted with LinfSkp1 in the co-IP assay (Fig 6C ), thus reinforcing the assumption of an indirect interaction between LinfFlp2 and LinfCul1 in its interactome.
To fully demonstrate the functional assembly of the active LinfCRL1(Flp1) complex, we developed a co-IP assay using anti-FLAG beads in HEK293T cells transfected with plasmids encoding LinfSkp1-FLAG, LinfCul1-myc, LinfRbx1-HA, and LinfFlp1-HA.The eluates confirmed the presence of the LinfCRL1(Flp1) complex with all components (Fig 6D).To further evaluate whether this complex was active, an in vitro ubiquitination assay was performed using human reagents E1(UBE1), E2 (UbcH5c/UBE2D3), and ubiquitin.We observed that LinfCRL1(Flp1) formed a polyubiquitination smear, confirming the activity of the purified complex (Fig 6E).
To visualize the F-box motif of Linf Flp1 interacting with LinfSkp1, Molecular Dynamics (MD) simulations of LinfCRL1(Flp1 1-100 ) were performed.The trajectory frames obtained in the MD were clustered in the UCSF Chimera [44], and a representative frame of the most populated cluster is shown (Fig 6F).The complex assembly is structurally conserved compared to that of H. sapiens (PDB ID:1LDK) [17] and Flp1-100 stably interacts with LinfSkp1.Interestingly, the MD simulation showed an intrinsic mobility of the LinfCul1 protein in a bending movement that brings LinfSkp1 and LinfRbx1 closer to each other.This movement can be observed by monitoring the distance between the centers of mass of the two proteins (Fig 6F, bottom and S1 Video).This analysis indicates that, at approximately 100 ns of simulation time, the distance between these proteins decreased to approximately 95 Å, increasing again to approximately 110 Å afterwards.This bending movement of LinfCul1 has not been previously described in its H. sapiens ortholog.

LinfCul1 is involved in proliferation and rosette formation
To evaluate whether LinfCRL1 proteins are essential for L. infantum promastigote survival, both alleles of the genes LINF_110018100 (SKP1), LINF_210005300 (RBX1), and LINF_240029100 (Cullin1) were simultaneously targeted for deletion using CRISPR-Cas9 technology [39].Only the Cullin1 knockout strain (Δcul1) was viable, indicating that LinfSKP1 and LinfRBX1 are potentially essential for L. infantum.In T. brucei, knockdown TbSkp1 and TbRbx1 by RNAi reduced the parasite proliferation rate, but both strains remained viable.Conversely, the knockout of TbCul1 did not lead to any observable effect, suggesting that other Cullins might perform its function in the parasite [45].The SKP1 and RBX1 genes in H. sapiens are associated with ubiquitination by CRL1 complexes, and these findings highlight the importance of this complex in the parasite.L. infantum Δcul1 was validated by PCR, which was used to determine the presence of the CUL1 gene coding sequence and the respective integration of the drug resistance markers (Fig 7A ).Growth curve analysis of the Δcul1 lineage compared with wild-type L. infantum and Cas9 T7 revealed that the LinfCUL1 knockout promoted a reduction in the growth rate from the fourth day onward (Fig 7B).To evaluate the cell growth rate more accurately, we performed a population doubling (PD) assay, in which we measured the cell line capacity to double over the course of culture days.In this assay, we assessed whether the proliferative potential of the cell line changed after cell passage.Interestingly, from the third passage forward, we found a divergence in the PD curves of the Δcul1 promastigotes compared to the respective controls, which was exacerbated over subsequent passages (Fig 7C).These findings indicated that LinfCUL1 is directly related to cell proliferation and affects the duplication capacity of the parasite.
Observations of cell cultures revealed a profile consisting of a high number of rosettes in the Δcul1 line compared to the wild-type strain, especially from the fourth day of growth (Fig 7D).Intriguingly, this structure coincided with the time point at which the proliferation rate decreased, indicating that they may be related.The images demonstrated an increase in both the number and size of rosettes over the course of culture (Fig 7E ), as well as intertwining flagella and the presence of fusion bodies (Fig 7F).Our findings indicated that LinfCUL1 likely regulates the formation of these structures.Nevertheless, it is unclear whether this effect is associated with the non-canonical function of LinfCul1 or with the LinfCRL1 complex.

LinfCul1 regulates the L. infantum cell cycle
The primary mechanism of cell cycle progression in eukaryotic cells is related to the successive activation of cyclin-dependent kinases (CDKs), which are mainly triggered by proteasomal degradation of their cyclin partners and kinase inhibitors (CKIs).Two eukaryotic E3 ubiquitin ligases, the anaphase-promoting complex (APC) and CRL1, are responsible for the ubiquitination of these regulators, thus regulating the progression of the cell cycle [24,46].Cul1 is the scaffold protein for the CRL1 complex that interacts with four different F-box proteins (FBXW7, βTrCP, SKP2, and Cyclin F), and mediates the ubiquitination and degradation of cell cycle key regulators, such as Myc, Cyclin F, CDK inhibitors (p21, p27 and p57), claspin, WEE1, EMI1 and REST [46].Here, we observed a reduction of cell proliferation in L. infantum Δcul1 compared to the parental cell lineage and carried out cell cycle analysis of both cell lineages to evaluate the function of LinfCul1 in this cellular process.
DNA content analysis by flow cytometry revealed a similar profile between Δcul1 and the parental cell lineage (Fig 8A , left).However, the overlap among the histograms revealed an accumulation of the cells in late S and G2/M phases in L. infantum Δcul1 (Fig 8A , middle).Analysis of cell distribution showed a significant reduction in 2n content (equivalent to G1/G0

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Cullin-1-RING ubiquitin-ligase in Leishmania infantum To investigate the progression of the S phase and infer the duration of the cell cycle phases, we used 5-ethynyl-2'-deoxyuridine (EdU) a thymidine analog to monitor DNA replication.For both lineages, EdU-labeled 2K2N parasites were first detected 0.75 h (45 min) after EdU addition, indicating that cells at the end of S phase required 45 min to proceed through G2 and M phases (Fig 8B).Next, we measured the proportion of EdU-labeled parasites after 1 h EdU pulse.Interestingly, L. infantum Δcul1 showed slightly more Edu-labeled cells than the parental cell lineage (Fig 8C).Also, both lineages showed a similar proportion of cells in cytokinesis (2K2N) (Fig 8D).Based on these data, S-phase duration was estimated using CeCyD software [47].We observed a significant increase in the duration of the S phase for the L. infantum Δcul1 lineage (S phase was estimated to be 2.751 h, or 0.24 ccu, for Δcul1 lineage, while the parental lineage presented an S phase equal to 0.78 h, or 0.1 ccu) (Fig 8E).Thus, our findings show that the increase in 4n content observed in Δcul1 lineage (Fig 8A) is actually cells accumulated in the late S phase, given that the end of this phase includes duplicated DNA content (4n), and its transition to G2 is not easily identified by flow cytometry.In other words, our results suggest that LinfCul1 regulates the S phase progression and possibly the transition between the late S to G2 phase in L. infantum through a mechanism that still needs to be investigated since there is no description of S/G2 checkpoint in trypanosomatids.

Discussion
Cullin-RING ubiquitin ligases (CRLs) mediate ubiquitination of protein substrates to regulate many aspects of eukaryotic biology, including cell division, signal transduction, transcription, metabolism, hormone perception, circadian rhythms, differentiation, and development [24].Many viruses and bacteria exploit CRLs activities to neutralize host antimicrobial defenses and facilitate their own replication [48].In Plasmodium berghei, CRL1(FBXO1) and SKP1/Cullin1/ FBXO1 (SCF FBXO1 ) complex are regulators of cell division and are essential for parasite-specific processes in the mammalian host and mosquito [49].T. brucei SKP1-Cullin-F-box (SCF) regulates cell cycle progression, where TbSkp1 participates in the G1/S transition, TbRbx1 in kinetoplast DNA (kDNA) replication, and the E2 CDC34 ubiquitin-conjugating enzyme is related to cytokinesis [45].As CRLs play crucial roles in a variety of organisms, we explored whether this class of E3 ubiquitin-ligases is conserved in Leishmania parasites.The orthologous genes of H. sapiens SKP1, CUL1, and RBX1 were conserved and were found in the Leishmania genus.Additionally, the amino acid residues responsible for SKP1, CUL1, and RBX1 interactions to assemble the CRL complex were conserved.This evolutionary conservation was corroborated by phylogenetic analysis, in which inferred phylogenies for SKP1, CUL1, and RBX1 appropriately reconstructed the known evolutionary relationships among Leishmania species.Notably, the large CUL1 protein is highly conserved across species.
The predicted three-dimensional structures of LinfSkp1, LinfCul1, and LinfRbx1 generated by AlphaFold [35,36], compared to their orthologs in H. sapiens, revealed a high degree of structural similarity in the high-confidence index region.The RMSD values suggest that LinfSkp1 closely resembles H. sapiens SKP1, whereas LinfCul1 and LinfRbx1 exhibit minimal structural disparities, which is interesting for selective drug development targeting LinfCRL1.
throughout the culture period.Images were captured using a 20X objective.F) Smear of the parental and L. infantum Δcul1 promastigote cultures after panoptic staining.Representative rosettes of each period were observed under an optical microscope at 100X objective.B-C) Graphs were generated using GraphPad Prism v5.0, and statistical significance was determined using a Two-Way ANOVA test (*p<0.05;***p<0.001).https://doi.org/10.1371/journal.ppat.1012336.g007

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Cullin-1-RING ubiquitin-ligase in Leishmania infantum  [47] was used to estimate the cell cycle phase duration.Representative images were obtained with 100X oil objective.EdU-labeled cells were revealed using azide conjugated with Alexafluor 488 (green).DAPI (blue) was used to stain DNA-containing organelles: kinetoplast (K) and nucleus (N).For A, three biological replicates were performed, and statistical significance was determined using the Strikingly, the in silico assembly of the LinfCRL1 complex superimposed with the human CRL1(SKP2) or SCF Skp2 crystal structure [17] demonstrated the same structural topology, with LinfCul1 working as a scaffold for the complex, binding to LinfSkp1 at its N-terminus and LinfRbx1at its C-terminus.To confirm these in silico data, co-IP assays were performed in HEK293T cells transfected with plasmids encoding the genes of interest.This mammalian cell line is highly transfectable and produce large amount of protein by culture being used as a platform to produce recombinant proteins [50].Co-IP results demonstrated that the conservation of interaction regions of LinfCRL1 proteins compared to H. sapiens was sufficient for their interactions and assembly of the complex.In addition, they revealed interactions among the components of L. infantum and H. sapiens, which might be explored by the parasite to evade the host immune system.Leishmania uses different survival strategies to evade or modulate host immune defense by regulating the ubiquitin proteasome system, which is responsible for parasite autophagy, DNA repair, and protein trafficking [51].L. donovani controls the deubiquitinating enzyme A20 to hinder TLR2-mediated proinflammatory gene expression, thereby evading host immune responses [52].Hepcidin-mediated degradation of ferroportin (Fpn) in macrophages is a widely adopted strategy to restrict iron availability to invading pathogens.L. donovani modulates the level of an F-box protein (FBXL5) in the host macrophage to activate IRE-IRP interaction, leading to the inhibition of Fpn.This inhibition promotes evasion of L. donovani from the immune system [53].Thus, our findings open perspectives for the exploration of host CRLs components by Leishmania infantum with consequences for the disease caused by the parasite.
To evaluate the assembly of the LinfCRL1 complex, the interactome of LinfSkp1 and Linf-Cul1 was acquired via immunoprecipitation followed by direct on-bead digestion and mass spectrometry protein identification.Remarkably, LinfCul1 and LinfRbx1 were identified in the LinfSkp1 interactome, and LinfRbx1 and LinfSkp1 were recovered from the LinfCul1 interactome confirming the presence of the LinfCRL complex in this parasite.Functional CRL1 complexes are conjugated to F-box proteins, which serve as specificity factors for this complex.These F-box proteins interact with SKP1 through the F-box domain and engage with their substrates through other domains [15][16][17].Interestingly, five F-box proteins (LinfFlp1, LinfFlp2, LinfFlp3, LinfFlp5, and LinfFlp6) were directly identified in the LinfSkp1 interactome, whereas LinfFlp4 was indirectly identified in the LinfCul1 interactome.The interaction of LinfFlp1 to 6 with LinfSkp1 was validated through co-IP assays, confirming the assembly of the LinfCRL1 complex with F-box proteins.Strikingly, an in vitro ubiquitination assay using in vitro purified LinfCRL(Flp1) demonstrated its capability to catalyze ubiquitin transfer.Molecular dynamic simulation of LinfCRL1 with the predicted F-box motif of Flp1(Flp1 1-100 ) demonstrated its interaction with LinfSkp1.However, whether this motif is sufficient for the interaction of Flp1 with LinfSkp1 needs to be studied.A bending movement of LinfCul1, bringing LinSkp1 and LinfRbx1 together, was also observed.Whether this conformational movement mediates the transfer of ubiquitin from E2 bound to LinfRbx1 to a substrate recruited by LinfSkp1 has to be explored.The human F-box proteins CRL(SKP2) (FBXL1) and CRL(β-TRCP) (beta-transducin repeat-containing protein) (FBXW1A) serve as primary regulators of the eukaryotes cell cycle [24].Additionally, various other CRL-associated Fbox proteins regulate a myriad of cellular processes in mammals [54], demonstrating their essential roles.This class of proteins remains unexplored in Leishmania spp.and only the TWO-WAY ANOVA test (*p<0.05;***p<0.001).For C and D, unpaired t-test was used (n = 3).Graphs were generated using GraphPad Prism v5.0 software.https://doi.org/10.1371/journal.ppat.1012336.g008

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Cullin-1-RING ubiquitin-ligase in Leishmania infantum F-box protein CFB2 has been studied in Trypanosoma brucei and described as required for cytokinesis of its bloodstream form [55]. Having shown their involvement in the assembly of LinfCRL1, investigation of their roles in these parasites is an intriguing area of investigation.We did not identify orthologs of H. sapiens F-box proteins in Leishmania spp., revealing poor sequence conservation in this class of proteins in these organisms, except for the expected conservation of the F-box domain.Consequently, we argue that LinfFlps are interesting targets for the development of selective anti-leishmanial drugs.
In the LinfSkp1 interactome, the presence of nucleic acid-binding proteins is noteworthy, with most of them being RNA binders, particularly the genes (LINF_260007900, LINF_320042200, LINF_330027700, LINF_320012500, LINF_330034300, and LINF_070016300).RNA-binding proteins (RBPs) in Leishmania spp.function as trans-regulators, overseeing the processing and trafficking of RNA molecules from synthesis to degradation.LINF_260007900 (Cleavage and Polyadenylation Specificity Factor Subunit 5) could potentially constitute a component of the cleavage factor Im (CFIm) complex, which acts as an activator in the pre-mRNA 3'-end cleavage and polyadenylation processes essential for the maturation of pre-mRNA into functional mRNAs.Five components of the Cleavage and Polyadenylation Specificity Factor complex (CPSF) were identified and confirmed through reciprocal co-immunoprecipitation in L. mexicana, which play a role in co-transcriptional cleavage and polyadenylation [56].Interestingly, the interactome of non-poly(A) RNA in L. mexicana revealed the presence of Skp1 and Cul1 [57], suggesting that the CRL complex may be involved in transcriptional regulation in Leishmania spp.In addition, LINF_180009700 (Paralyzed flagella protein 20/Small ribosomal subunit protein RACK1/ Guanine nucleotide-binding protein subunit beta-like protein) could be part of the core ribosomal proteins that repress gene expression and is involved in the induction of the ribosome quality control (RQC) pathway [58,59], which degrades nascent peptide chains during problematic translation [60].Intriguingly, the majority of the LinfCul1 interactome comprises ribosomal proteins, indicating a correlation between CRL1 with translational regulation and RQC in Leishmania spp.
The formation of H. sapiens CRL1 complexes is governed by the covalent attachment of a ubiquitin-like protein, namely NEDD8, at amino acid residue 720 (K720) in the C-terminus of Cullin1.Neddylation is a process akin to ubiquitination, involving the E1, E2, and E3 classes of enzymes.This process is hindered by the binding of CAND1 (cullin-associated and neddylation-dissociated 1) protein to Cullins, inhibiting complex assembly.The specific isopeptidase activity of the COP9 signalosome (CSN) complex facilitates the removal of NEDD8 from Cullins, thereby enabling the binding of SKP1-F-box proteins to the CRL complex [61].The E1 enzyme responsible for neddylation (UBA3) and the NEDD8-transferring enzyme UBC12 have been identified in the L. mexicana proteome [19] with their respective orthologs, LINF_010012200-T1 and LINF_240023100-T1, found in L. infantum.Here, we identified in LinfSkp1 and LinfCul1 interactome the gene LINF_340026400 (ubiquitin-like protein), which has not been explored.The BLAST search of this gene in UniprotDB identified the NEDD8 protein in different species of Fusarium fungus or the Ubiquitin-NEDD8-like protein RUB2 in Nicotiana tabacum.We speculated that the ubiquitin-like protein identified in the LinfCul1 interactome may serve as a regulator of the L. infantum CRL complex, functioning similarly to NEDD8 in H. sapiens.As expected, this protein was also present in the LinfSkp1 interactome, as the assembly of CRL requires the conjugation of NEDD8 to CUL1.Consequently, Linf-Cul1-NEDD8 co-eluted with LinfSkp1 in its interactome.Interestingly, the K720 residue is also conserved in Leishmania spp.CUL1, supporting the hypothesis that CRL1 is regulated by neddylation in these parasites.
The knockout of LinfSKP1 and LinfRBX1 results in nonviable L. infantum strains, demonstrating that they have essential roles in this parasite.Knockout of LmxM.31.0450 or LmxM.36.5820, two orthologous genes of LinfSKP1 and LinfRBX1 in L. mexicana, respectively, led to nonviable and viable parasites (LeishGEM data browser: https://browse.leishgem.org/).These outcomes may stem from the varied functions of RBX1 across different Leishmania species or from potential functional compensation upon knockout in L. mexicana.In T. brucei, TbRBX interacts with five distinct Cullins: TbCul-A, TbCulC, TbCulD, TbCulE, and TbCulF.This finding suggests that TbRBX plays various roles associated with multiple Cullins, underscoring its essential function in this parasite [62].Conversely, SKP1 deletion resulted in nonviable lineages in both species, emphasizing the crucial role in these parasites.L. infantum Δcul1 was viable, but the strain exhibited impaired cell proliferation and an increased doubling time, suggesting that LinfCul1 plays some role in regulating the cell cycle progression.Interestingly, according to DNA content analysis and the EdU incorporation assay (Fig 8), LinfCul1 is possibly involved in S phase progression and in regulating the transition from the late S to G2 phase in this parasite, although there are no known control mechanisms for the S/G2 transition in trypanosomatids to date.In T. brucei, no phenotypic changes were observed after the knockout of TbCUL1, suggesting that it might be functionally redundant with other Cullins [45].Whether the cell cycle effect of LinfCul1 is related to its function as a LinfCRL1 component or a non-canonical function has to be determined, since other CRL1 components have atypical functions such as SKP1 and F-box proteins [63,64].In L. donovani, the cell cycle is regulated by the Cdc20 protein (LDCdc20p), a regulator of the Anaphase Promoting Complex/Cyclosome (APC/C), which mediates ubiquitin-dependent proteasomal degradation of key cell cycle regulators in eukaryotes [65].CRL1 and APC/C are structurally similar E3 ubiquitin ligases that jointly regulate the cell cycle.While APC/C is active from mid-mitosis (anaphase) to the end of G1 phase, the CRL1 complex is functionally active from late G1 to early M phase [24].Interestingly, L. infantum exhibits nine orthologous genes in the H. sapiens APC/C complex (LINF_350050800, LINF_350042900, LINF_240023200, LINF_300024800, LINF_050009100, LINF_300012300, LINF_300035500, LINF_120011300, and LINF_040009000), implying that the cell cycle may be regulated by the CRL1 and APC/C complexes.
The L. infantum Δcul1 line showed parasite agglomeration as promastigotes, reminiscent of rosette formation.These structures have been described in L. major expressing polysialic acid (PSA) on the cell surface as well as acetylated neuraminic acid (NeuPSA) contained within PSA.The frequency of rosette formation and the expression of PSA/NeuPSA on the cell surface were temperature dependent.Rosettes form structures resembling cell fusion bodies that are then released.It is hypothesized that rosettes represent an unrecognized phase in the life cycle of Leishmania spp., starting the mating process, during which the expression of PSA/ NeuPSA plays a crucial role in L. major [66].In addition to temperature stress, rotational movement of the cell culture also influences the formation of cellular aggregates, similar to rosettes [67].The structures observed in the Δcul1 strain resemble those observed in L. major, with intertwining flagella and fusion bodies.The initial observation of potential sexual reproduction in Leishmania was derived from quantitative microspectrophotometry, suggesting the occurrence of nuclear fusion or sexual reproduction within the intracellular amastigote form [68].The identification of hybrid genotypes in natural isolates of Leishmania has further supported this hypothesis, and computational methods have been employed to investigate aneuploid genome dynamics [69].Genetic exchange in Leishmania can be facilitated by IgM antibodies, which induce the formation of spherical parasite clumps, thereby promoting the fusion of parasites and formation of hybrids [70].HOP1 and a HAP2 paralog (HAP2-2) have been shown to be essential components of the Leishmania meiosis machinery and the cell-tocell fusion mechanism associated with sexual reproduction in these parasites [71].Our results demonstrate that knockout of LinfCUL1 stimulates the formation of rosettes, indicating that the expression of this gene represses or reduces the formation of these structures.The Cullin protein family has been reported to be associated with the regulation of meiosis and chromosome segregation in different organisms.Cullin9 protects mouse eggs from aneuploidy by controlling microtubule dynamics during oocyte meiosis [72], and Cullin 4A regulates meiotic progression in mouse spermatogenesis [73].SCF/Skp1 together with Fbh1 ensures proper chromosome segregation in fission yeast [74].In Caenorhabditis elegans, CUL-2 controls various crucial processes in cell division and embryonic development, including meiotic progression, anterior-posterior polarity, and mitotic chromatin condensation [75].Whether CUL1 or the CRL1 complex is responsible for the division of fused parasites during sexual reproduction of Leishmania remains to be explored.
In summary, we demonstrated that the L. infantum genes LINF_110018100 (SKP1-like protein), LINF_240029100 (cullin-like protein), and LINF_210005300 (putative ring-box protein 1) form a LinfCRL1 complex in combination with six F-box proteins (Flp1-6), potentially regulated by a NEDD8-like protein LINF_340026400.The LinfCRL1 complex, when conjugated to Flp1, exhibited activity in in vitro ubiquitination assays.LinfSkp1 and LinfCul1 interactomes revealed proteins associated with diverse cellular processes, suggesting their roles in transcription and translation, respectively.The knockout of LinfSKP1 and LinfRBX1 produced nonviable lineages, whereas L. infantum Δcul1 was viable, with an impaired proliferation rate, cell cycle phase alterations, and stimulation of rosette formation.Thus, we characterized Cul-lin1-RING ubiquitin ligases or SCF1 in L. infantum, as well as their protein partners.These findings not only advance our understanding of the molecular machinery governing Leishmania biology but also provide potential targets for therapeutic intervention against visceral leishmaniasis.

Parasite cultures and proliferation
The Leishmania infantum isolate HU-UFS14, characterized by experimental infections [76][77][78] and whole-genome sequencing [79], was used.Promastigotes were cultivated and maintained weekly at 27˚C in Schneider's Drosophila Medium (Gibco) supplemented with 20% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin/L-glutamine solution (Gibco), and 2% filtered male urine [80].Promastigotes at a density of 1 × 10 5 cells/mL were added to 12-well plates in triplicate, and an aliquot from each well was counted daily for 9 days to determine the growth.

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Cullin-1-RING ubiquitin-ligase in Leishmania infantum Duplication rate (DR) or population doubling (PD) assays were carried out as [81], and 1 × 10 5 cells/mL were cultivated in M199 supplemented with the antibiotics mentioned above.Each strain was cultivated in 12 wells plate and the cell number was determined daily, and the same number of cells were seeded per well for 8 consecutive days.PD was determined as Log 2 [TCN/(2 × 10 5 ) per day] = growth rate (GR) (TCN = Total Cell Number).Thus, the GR on day 2 (GRD2) was GRD1+GRD2, and GRD3 = GRD2+GRD3, among others.The values plotted on the graph are the means of triplicates for each time point for each day, and two-way ANOVA was used in GraphPad Prism v5.0.

Immunoprecipitation of LinfSkp1 and LinfCul1
Three independent replicates of L. infatum 3x myc-mCherry::SKP1 or L. infatum 3x myc-mCherry::CUL1 and two independent replicates of L. infantum Cas9 T7 as a negative control were used.For immunoprecipitation, 4 × 10 9 parasites were washed twice in PBS and lysed with cold NP-40 lysis buffer (50 mM Tris-HCl pH 7.2, 225 mM KCl, and 1% NP-40) supplemented with a protease inhibitor cocktail SIGMAFAST (Sigma Aldrich) and phosphatase inhibitors (10 mM NaF and 1 mM Na 3 VO 4 ) with three cycles of freezing in liquid nitrogen and thawing at room temperature.Later, the lysates were maintained on ice for 30 min and then centrifuged for 20 min at 14,000 × g at 4 ˚C.The supernatant protein concentration was determined using Bradford (Sigma Aldrich).For each immunoprecipitation, 20 mg of total protein was incubated with 60 μL of anti-myc beads (Sigma Aldrich, #E6654) for 3 h at 4 ˚C with rotation.Beads were washed with 5 × 500 μL ice-cold lysis buffer and 3 × 500 μL ice-cold wash buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl 2 , 225 mM KCl) following on-bead digestion [40].

Sample preparation for mass spectrometry analyzes
Trypsin digestion was performed by resuspending beads in 93 μL of 50 mM ammonium bicarbonate and 1 μL of 0.5 M dithiothreitol (DTT), and the samples were incubated at 56˚C for 20 min.Next, 2.7 μL of fresh 0.55 M iodoacetamide was added, and the samples were incubated at room temperature for 15 min protected from light.That, 1,3 μL of trypsin gold, mass

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Cullin-1-RING ubiquitin-ligase in Leishmania infantum spectrometry grade (Promega V5280), was added, and the samples were mixed and incubated overnight at 37˚C.The reaction was stopped by the addition of 1 mL of trifluoroacetic acid (liquid chromatography-mass spectrometry [LC-MS] grade), and incubated for 5 min [40].Then, the samples were desalted using Stage Tips with C18 membranes (Octadecyl C18-bonded silica -3M Empore extraction disks) and completely dried in an evaporator (SPD 1010 SpeedVac, Thermo).

Analysis by Nanoflow nLC-MS/MS
The samples were reconstituted in 10 μL of formic acid, and an aliquot of 1 μL was analyzed using an ETD-enabled Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) coupled to an EASY-nLC system (Proxeon Biosystem, West Palm Beach, FL, USA) via a Proxeon nanoelectrospray ion source.Peptides were separated using an analytical column, PicoFrit Column (20 cm × ID75 μm, 5 μm particle size, New Objective), employing a 2-90% acetonitrile gradient in 0.1% formic acid at a flow rate of 300 nL�min−1 over a 65-minute duration.The nanoelectrospray voltage was set to 2.2 kV, and the source temperature was maintained at 275 ˚C.The instrument methods were configured in a data-dependent acquisition mode.Full-scan MS spectra (m/z 300-1600) were acquired using the Orbitrap analyzer after accumulation to a target value of 1 × 10 6 .The resolution in the Orbitrap was set to r = 60,000, and the 20 most intense peptide ions with charge states �2 were sequentially isolated to a target value of 5,000 and fragmented in the linear ion trap using low-energy CID (normalized collision energy of 35%).The signal threshold for triggering an MS/MS event was established at 1,000 counts.Dynamic exclusion was activated with an exclusion size list of 500, an exclusion duration of 60 s, and a repeat count of 1.An activation of q = 0.25 and activation time of 10 ms were applied [84].

Raw LC-MS/MS data analysis
Protein identification was conducted using Proteome Discoverer version 1.4 (Thermo Fisher Scientific), employing the Sequest search algorithm.The analysis was performed using the Leishmania infantum protein database (release 2022; 8272 sequences; 5263968 residues) obtained from UniprotDB.Carbamidomethylation was designated as a fixed modification, whereas methionine oxidation was considered a variable modification.Parameters for protein identification included a maximum of two trypsin missed cleavages and tolerance levels of 10 ppm for precursor mass and 1 Da for fragment ions.A filtering criterion was applied to maintain a maximum false discvery rate of 1% at both peptide and protein levels.Proteins identified in at least one negative control were excluded from those identified in the SKP1 and Cul1 interactomes.Proteins identified with at least one unique peptide present in all three samples of each interactome in triplicate were considered hits and are listed in S2 Table.

Phylogenetic analysis
Nucleotide sequences of each human gene were used to search for orthologs in 12 Leishmania species (L.braziliensis, L. panamensis, L. amazonensis, L. mexicana, L. donovani, L. infantum, L. aethiopica, L. tropica, L. major, L. gerbilli, L. arabica, and L. turanica) using blastx in Tri-TrypDB [85] (Target Data Type: Proteins; BLAST Program: blastx; Target Organism: Leishmania).The sequences of SKP1, CUL1, and RBX1 human proteins and their orthologs in the 12 Leishmania species are listed in S1 Table.They were subjected to multiple sequence alignment (MSA), and the most appropriate amino acid substitution model was determined for each gene.CUL1 and RBX1 used the JTT matrix [30] and SKP1 used Le Gascuel [29] as an evolutionary model of amino acid substitution.For RBX1, a discrete gamma distribution was

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Cullin-1-RING ubiquitin-ligase in Leishmania infantum used to model evolutionary rate differences among sites (five categories (+G, parameter = 0.0575)).Phylogenetic reconstruction of each gene was performed with Maximum Likelihood (ML) method [86] using MEGA (version 11) and Bayesian Inference (BI) method using BEAST (version 1.10.4)[33].For ML method the reliability of the obtained phylogeny was subsequently evaluated using the statistical bootstrap method [87], using a minimum of 1000 replicates.The bootstrap method generates data replicates from which multiple phylogenetic trees can be constructed.The bootstrap value associated with each branch of the tree reflects, as a percentage, the frequency with which a specific clade was observed in the replicates, indicating higher reliability in the phylogenetic relationships for higher values.For BI method, starting tree was random and molecular clock was strict as default [88].Tree prior distribution was set to Yule speciation [89].For each gene was performed 10 7 iterations and sampling every 10 4 iterations.For burn-in was set 10% of the iterations.Maximum Clade Credibility (MCC) trees were generated using TreeAnnotator using median heights for each gene.BI-inferred trees were visualized in FigTree (available at http://tree.bio.ed.ac.uk/software/figtree/).

Protein structure modeling
The structures of the proteins encoded by genes the LINF_110018100, LINF_240029100, and LINF_210005300 from Leishmania infantum (JPCM5) were obtained from AlphaFold database [35,36] and subsequently compared to human orthologs of the CRL1 complex [17].Amino acid residues were colored according to the reliability of their structure: dark blue for high reliability, light blue for moderate reliability, yellow for low reliability, and orange for very low reliability.The prediction of the CRL1-type E3 ubiquitin ligase complex was performed using the AlphaFold2 (AF2) (AlphaFold v2.3.2 online version) [35].The obtained models were visualized and analyzed using UCSF Chimera software (version 1.17.3)[90].RMSD was calculated using the DALI server, a server for comparing protein structures in 3D [91].For identical structures, the RMSD is 0, and values between 0 and 2 Å indicated a high percentage of identity [37].

Molecular Dynamics (MD) simulation
The AF2 implemented in ColabFold [92] was used for generation of LinfCRL1 complex structure including the F-box motif of Flp1 .This model were used for the MD simulations using the AMBER 22 package [93][94][95].The protonation states of the residues were defined using PDB2PQR [96].The AMBER FF19SB force field was employed along with the OPC water model.The system was immersed in a cubic box filled with water, Na + , and Cl − to neutralize the system and maintain a salt concentration of 150 mM.The system energy was minimized in 8,000 steps of energy minimization, followed by heating to 300 K in 100 ps of the NVT simulation.The system density was then equilibrated by 100 ps of NPT simulation, followed by a 2 ns final equilibration in the NPT ensemble.A 250 ns simulation of the equilibrated system in the NPT ensemble was used for analysis.The entire system containing approximately 850,000 atoms was simulated using the hydrogen mass repartition scheme [97] and a 4 fs timestep.Analysis of the obtained trajectory file was performed using CPPTRAJ [98] and AmberEnergy++ [99].

In vitro ubiquitination assay
HEK293T cells were transfected with LinfCRL1 (Flp1) and rinsed with the lysis buffer.The supernatants were immunoprecipitated using anti-FLAG M2 beads, and the complex was eluted with 100 μg/ml of 3XFLAG peptide (Sigma-Aldrich) in Tris-buffered saline (TBS; 50mM Tris-HCl, pH 7.5, 0.15 M NaCl).The in vitro ubiquitination reactions were developed

Rosette formation analysis
The cell lines were seeded in triplicate in 25 cm 2 culture flasks containing 2 x10 5 cells/mL in 5 mL of complete M199 supplemented with 0,1 mM adenine, 2,5 mg/ml hemin, 1% penicillinstreptomycin, and 20% FBS, with the respective selection antibiotics added.Triplicate flasks for each cell line were observed, photographed, and filmed per field (four corners of the flask and its central portion, totaling five fields) every 24 h for 7 days, using OPTIKA PROVIEW v4.11 software via the C-B5 OPTIKA Microscopes ITALY camera, with a magnification of 20X.Data were obtained from the analysis of five regions, including the four corners and central portion of the culture flask.The rosette count was normalized to the number of photographed fields per culture flask, and the values were plotted.Statistical differences and graph generation were assessed by two-way ANOVA variance using GraphPad Prism v5.0.To analyze the structure of rosette formation at higher magnification, a slide smear was prepared using the L. infantum Δcul1 strain.The samples were stained using the Romanowsky technique (Laborclin) and left to dry for 24 h.After this period, the slides were analyzed using an Olympus BX50 optical microscope with a 20X or 100X objective, and images were generated by the DP2-BSW v.2.2 software using the Olympus DP72 camera software.

Flow cytometry
Promastigotes of L. infantum Cas9 T7 and L. infantum Δcul1 were cultivated at a concentration of 2 x 10 7 cells/ml.On the 2 nd day of the growth curve, the cells were harvested by centrifugation (1000 x g at 4 ˚C for 5 min) and washed in PBS at 4 ˚C.Subsequently, the cells were resuspended in 70% methanol (diluted in PBS) and incubated overnight at 4 ˚C.After this period, the cells were washed twice, and the pellet was resuspended in PBS containing propidium iodide (PI) (10 μg/ml) and RNase A (10 μg/ml) and incubated for 30 min at 37 ˚C.The parasites were analyzed using a flow cytometer (Attune Acoustic Focusing Flow Cytometer-Thermo Scientific).For each sample, 50,000 events were analyzed.The variance between these groups was similar.Images were drawn up using FlowJo v.7.6.5 software (FlowJo, LLC, Ashland, OR).

Cell cycle analysis by EdU incorporation
Formaldehyde-fixed and DAPI-stained exponentially growing promastigotes were examined under a fluorescent microscope (Axiovert 200M -Zeiss), (100X oil objective) to observe the profile of organelles that contain DNA (nucleus and kinetoplast).The profile 2K2N was used to estimate the percentage of cells undergoing cytokinesis (C).Using this parameter for the samples analyzed, we estimated the duration of cytokinesis according to the Williams Equation [100]: where x is the cumulative time within the cycle until the end of the stage in question, y is the

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Cullin-1-RING ubiquitin-ligase in Leishmania infantum cumulative % of cells up to and including the stage in question (expressed as a fraction of one unit), and α is the specific growth rate.To estimate the G2+M phase length, we added EdU to the medium containing exponentially growing promastigotes and collected samples every 15 min, proceeding with 'click' chemistry reaction, until a parasite containing two EdU-labeled nuclei (2K2N) was observed (corresponding to the length of G2+M phases).To determine the S-phase duration, we measured the proportion of EdU-labeled cells after 1 h EdU pulse.The Sphase duration was estimated according to the Stanners and Till Equation (Stanners and Till 1960) [101]: where L is the proportion of cells exhibiting EdU-labeled nuclei, α = ln 2.T -1 (T = doubling time, expressed in h), Z = G2 + M + cytokinesis, and t is the duration of the EdU pulse in hours (h).Finally, the duration of the G1 phase was estimated by the difference between the doubling time (dt) and the sum of the other phases (S+G2+M+C).Of note, all these calculations were made with the help of the online software CeCyD, available at the address https:// cecyd.vital.butantan.gov.br/[47].

Fig 2 .
Fig 2. Phylogenetic trees of CRL1 complex proteins from Leishmania.A) CUL1, B) RBX1 and SKP1 (C-D) cladograms built with 12 orthologs of Leishmania spp.(accession numbers in S1 Table).The proteins were aligned using the MUSCLE method[27] and trees were reconstructed using ML and BI methods.A total of 183, 744, and 106 positions for SKP1, CUL1, and RBX, respectively, were considered for the final dataset.ML analysis was performed using MEGA11 software[32], while BI analysis was performed using BEAST software[33].The numbers next to the branches represent the statistical support of phylogenetic inference displayed by method side by side, IB (probability) / ML (boostrap), for CUL1 (A) and RBX1 (B); for SKP1 cladograms are displayed according to the method of inference (C and D).Species within Leishmania and Viannia subgenera are indicated in blue and red font, respectively.

Fig 6 .
Fig 6.Characterization of LinfFlps and molecular dynamics of LinfCRL1(Flp1 1-100 ).A) F-box consensus sequencing alignment with the F-box domain of Flps 1-6, where boxes represent the most conserved amino acid residues.B) To validate the LinfSkp1 interactome, HEK293T cells were transfected with the indicated plasmids, and cellular extracts were subjected to immunoprecipitation with anti-FLAG agarose beads.Co-immunoprecipitation of LinfSkp1 with LinfFlp1, LinfFlp2, LinfFlp3, LinfFlp5, and LinfFlp6 (B), and LinfFlp4 (C).LinfCRL1(Flp1) was purified (D) and its activity was evaluated using an in vitro ubiquitination assay (E).F) Molecular dynamic simulation of LinfCRL1(Flp1 1-100 ), where LinfCul1 is shown in blue cartoon, LinfSkp1 is shown in orange cartoon while LinfRbx1 and LinfFlp1 1-100 are shown in green and pink cartoon, respectively.The pcDNA3.1 plasmid is an empty vector utilized as a negative control.Antibodies used are shown.These assays were performed in duplicate.https://doi.org/10.1371/journal.ppat.1012336.g006

Fig 7 .
Fig 7. Effects of LinfCUL1 knockout on L. infantum promastigotes.A) Amplicons from L. infantum HU-UFS14, Cas9 T7, and Δcul1 lines generated by PCR with the primers shown in the right diagram, which depicts the LinfCUL1 gene locus and primer annealing sites (arrows).B) Growth curves of wild-type L. infantum, the Cas9 T7 transgenic line, and Δcul1.Cell counts were performed every 24 h for 8 days.C) Cell proliferation rate analysis by population doubling over eight days of culture between the control and Δcul1 strains.D) Comparison of rosette formation between the control and Δcul1 strains over an 8-day culture period.E) Promastigote culture images of the parental and Δcul1 strains showing rosette formation and growth

Fig 8 .
Fig 8. Cell cycle analysis of L. infantum Δcul1 and the parental cell line.A) Flow cytometry analysis and quantification of DNA content.B) Estimation of the duration of the G2+M phases through the minimum time to find two EdU-labeled nuclei in the same cell.C) Percentage of EdU-labeled cells after 1 h EdU pulse.D) DAPI-labeled parasites with 2K2N configurations were used to estimate the percentage of cells performing cytokinesis.E) The CeCyD software[47] was used to estimate the cell cycle phase duration.Representative images were obtained with 100X oil objective.EdU-labeled cells were revealed using azide conjugated with Alexafluor 488 (green).DAPI (blue) was used to stain DNA-containing organelles: kinetoplast (K) and nucleus (N).For A, three biological replicates were performed, and statistical significance was determined using the